Prior Art
In the last twenty years, a wide variety of agents have been used as labels in clinical diagnostic and research assays. More recently, hybridization assays have been developed for sensitively detecting the presence of unique polynucleotide sequences. Typically, in such assays, a nucleotide multimer (probe) is labelled with an atom or group which can be readily detected.
When the labelled probe is exposed to a test sample suspected of containing a target nucleotide sequence, under hybridizing conditions, the target will hybridize with any such labelled probe. The presence of the target sequence in the sample can be determined qualitatively, or quantatively usually by separating hybridized and non-hybridized probe, then determining the amount of labelled probe which hybridized, either by determining the presence of label in probe hybrids, or by determining the quantity of label in non-hybridized probes.
Historically, radioactive labels were used. However, due to health hazards and difficulties in handling, non-isotopic labels were later developed. Such labels include those whose presence is determined either directly or indirectly. Examples of direct labels include chemiluminescent, fluorescent, or spectroscopically detectable labels. Examples of indirect labels include compounds, such as biotin and various antigens, which can be detected by means of proteins conjugated to an appropriate detectable label.
One preferred method for introducing labels into nucleotide multimer probes has been to introduce linker-arms into enzymatically or chemically synthesized probes. For example, 4-thio-UTP (H. Eshaghpour et al., Nucl. Acids Res., Vol. 7, p. 1485, 1979) has been attached to the 3'-end of DNA fragments and subsequently labelled at its nucleophilic sulfhydryl moiety. Another method disclosed in a PCT application by Tchen (International Publication No. WO 86/00074; published Jan. 3, 1986) disclosed a technique in which pyrimidine base nucleotides are depyrimidated, and the resulting sugar rings opened so that an amine bearing moiety can be attached thereto.
In addition, the 5-allylamine uridine triphosphate precursor analogs disclosed by P. R. Langer et al. (Proc. of Nat. Acad. Sci., U.S.A., Vol. 78, p. 6633, 1981), may be used for incorporating nucleophilic amines moieties into nucleotide multimer probes which provide sites for labelling.
Chemical methods for labelling have also been proposed which allow labels to be linked to nucleotides in a nucleotide multimer. One such method involves bisulfite catalyzed transamination with ethylenediamine at the C-4 position of cytosine residues of nucleic acid probes (R. P. Viscidi et al., J. Clin. Biol., Vol. 23, p. 311, 1986). Other techniques have been disclosed which allow attachment of only a single label at the 5'- or 3'-end of a nucleotide multimer, typically an oligonucleotide. For example, terminal labelling approaches have been disclosed which allow linker-arm attachment as a final step in solid-phase oligonucleotide synthesis. Such linker-arms are then employed for label attachment. For example, see B. A. Connolly, Nucl. Acids Res., Vol. 13, p. 4485, 1985; S. Agrawal et al., Nucl. Acids Res., Vol. 15, p. 3131, 1987.
Compounds have also been disclosed which can be used to insert a primary amine-modified nucleotide residue at selected positions in a synthetic oligonucleotide during standard automated synthesis procedures. Such compounds include analogs of deoxythymidine and deoxyadenine, deoxyguanine, etc. (G. B. Dreyer et al., Proc. Natl. Acad. Sci., U.S.A., Vol. 82, p. 968, 1985; J. L. Ruth, PCT application No. US84/00279, Publication No. WO 84/03285, published Aug. 30, 1984). In addition, alkylamine derivatives of nucleotide linking phosphate groups have been disclosed, the amino functional group of which can then be labelled (R. L. Letsinger and M. E. Schott, Jour. Amer. Chem. Soc., Vol. 103, p. 7394, 1981; Japanese patents to N. Sugimoto Nos. 61 44,353 issued Mar. 4, 1986; 61 57,595 issued Mar. 24, 1986; 61 44,352 issued Mar. 4, 1986). Theoretically, such compounds might allow for labelled nucleotides to be placed at a number of sites along a sequence, thus permitting use of multiple labels to increase sensitivity of detection. However, one must be careful in selecting linker-arm locations since some linker-arm sites can reduce the stability of a hybrid formed with a target sequence, particularly when multiple labels are present.
In addition to the linker-arms described above, we have designed non-nucleotide based linker-arm reagents which are disclosed in two pending patent applications to Arnold et al. entitled "Non-Nucleotide Linking Reagents for Nucleotide Probes" U.S. application Ser. No. 099,050 filed Sep. 21, 1987, now abandoned and "Non-Nucleotide Reagents for substituting the Termini of Oligonucleotides" U.S. application Ser. No. 104,330 filed Oct. 2, 1987, now abandoned. These linker-arm reagents overcome limitations of other prior-art reagents and allow attachment of linker-arms at a wide variety of sites as well as allowing the construction of nucleotide/non-nucleotide polymers.
One of the most sensitive classes of non-isotopic labels known are the chemiluminescent acridinium esters which have been described by Campbell et al., in U.K. patent No. 2112779B issued Oct. 15, 1986 and in Richardson et al., Chemiluminescence Immunoassay of Plasma Progesterone, with Progesterone-Acridinium Ester Used As the Labeled Antigen, Clin. Chem., Vol. 31, pp. 1664-1668 (1985), for attaching acridinium esters to proteins and hormones for use in immunodiagnostic assays. The attachment of acridinium esters to nucleotide probe multimers is not readily achieved using the labelling and purification procedures described for proteins. Labeling of nucleotide multimer probes with acridinium esters have been suggested earlier. However, such suggestions provide no method for labelling and purification (Yabusaki et al., U.S. Pat. No. 4,599,303) or the procedures described give poor extents of labelling and inadequate degrees of purification of labelled probes for use in hybridization assays (Mock et al., EPA Application No. 86306305.3 filed Aug. 15, 1986, Publication No. 0212951). The method described by Mock and Septek, for example, describes a procedure analogous to that for labelling and purifying proteins (Weeks et al.). We have found that such methods are inadequate for labelling oligonucleotide probes containing amineterminated linker-arms to any significant extent (.ltoreq.10%). Moreover, the gel filtration methods employed for purifying acridinium ester labelled proteins do not separate labelled and unlabelled probe nor do they adequately remove unconjugated label. For use in hybridization diagnostic assays, it is necessary that neither significant amounts of unlabelled probe be present, since such unlabelled probes will compete in hybridization reactions with labelled probes, nor that the purified sample consists of more than 1% unconjugated label since unconjugated label binds to separation supports and creates high chemiluminescent background which greatly reduces the sensitivity of diagnostic assays. What is needed are means for labelling nucleotide multimers and purifying labelled multimers in essentially a pure form. The invention described herein provides such means.